1. Field of the Invention
This invention relates to rolling circle amplification processes that incorporate nucleotide analogs (“non-standard nucleotides”) that form base pairs joined by hydrogen bonding patterns not found in standard nucleotides A, T, G and C.
2. Description of Related Art
Natural oligonucleotides bind to complementary oligonucleotides according to well-known rules of nucleobase pairing first elaborated by Watson and Crick, where adenine (A) pairs with thymine (T) (or uracil, U, in RNA), and guanine (G) pairs with cytosine (C), with anti-parallel complementary strands. In this disclosure, “DNA”, “oligonucleotide”, or “nucleic acid” is understood to include DNA and RNA, as well as derivatives where the sugar is modified, as in 2′-O-methyl and 2′,3′-dideoxynucleoside derivatives, where the nucleobase has an appendage, and these nucleic acids and their analogs in non-linear topologies, including as dendrimers, comb-structures, and nanostructures, and analogs carrying appendages or tags (e.g., fluorescent, functionalized, or binding, such as biotin). Further, “polymerase” in this application is meant to include DNA polymerases of all families, RNA polymerases, and reverse transcriptases.
These pairing rules allow specific hybridization of oligonucleotides to complementary oligonucleotides, making oligonucleotides valuable as probes in the laboratory, in diagnostics, as messages that direct the synthesis of proteins, and in other applications known in the art. Such pairing is used, for example and without limitation, to capture oligonucleotides to beads, arrays, and other solid supports, allow nucleic acids to fold in hairpins, beacons, and catalysts, support function, such as fluorescence, quenching, binding/capture, and catalysis, and as part of complex structures, including dendrimers and nanostructures, and scaffolds to guide chemical reactions.
Further, base pairing underlies the enzymatic synthesis of oligonucleotides complementary to a template. Here, assembly of building blocks from nucleoside triphosphates is directed by a template to form a complementary oligonucleotide with a complementary sequence. This is the basis for replication in living systems, and underlies technologies for enzymatic synthesis and amplification of specific nucleic acids by enzymes such as DNA and RNA polymerase, the polymerase chain reaction (PCR), and assays involving synthesis, ligation, cleavage, immobilization and release, inter alia.
Watson-Crick pairing rules can be understood as the product of two rules of complementarity: (1) size complementarity (a big purine pairs with a small pyrimidine) and (2) hydrogen bonding complementarity (hydrogen bond donors pair with hydrogen bond acceptors). However, as noted by U.S. Pat. Nos. 5,432,272, 5,965,364, 6,001,983, 6,037,120, 6,140,496, 6,627,456, and 6,617,106, Watson-Crick geometry can accommodate as many as 12 nucleobases forming 6 mutually exclusive pairs. Of these, four nucleobases forming two pairs are designated “standard”, while eight nucleobases forming four pairs were termed “non-standard”, and may be part of an “artificially expanded genetic information system” (AEGIS).
To systematize the nomenclature for the hydrogen bonding patterns, the hydrogen bonding pattern implemented on a small component of a nucleobase pair are designated by the prefix “py”. Following this prefix is the order, from the major to the minor groove, of hydrogen bond acceptor (A) and donor (D) groups. Thus, both thymine and uracil implement the standard hydrogen bonding pattern pyADA. The standard nucleobase cytosine implements the standard hydrogen bonding pattern pyDAA. Hydrogen bonding patterns implemented on the large component of the nucleobase pair are designated by the prefix “pu”. Following the prefix, hydrogen bond donor and acceptor groups are designated, from major to minor groove, by “A” and “D”. Thus, the standard nucleobases adenine and guanine implement the standard hydrogen bonding patterns puDA- and puADD respectively.
A central teaching of this disclosure is that hydrogen-bonding patterns are distinct from the organic molecule that implements them. Thus, guanosine implements the puADD hydrogen-bonding pattern. So does, however, 7-deazaguanosine, 3,7-dideazaguanosine, and many other purines and purine analogs, including those that carry side chains carrying functional groups, such as biotin, fluorescent, and quencher groups. Which organic molecule is chosen to implement a specific hydrogen-bonding pattern determines, in part, the utility of the non-standard hydrogen-bonding pattern, in various applications to which it might be applied.
As described by U.S. Ser. No. 12/999,138, which is incorporated in its entirety by reference, claims of U.S. Pat. No. 5,432,272 and its successors covered non-standard bases that implemented the pyDDA hydrogen bonding pattern that encountered problems, including epimerization, oxidation, and uncharacterized decomposition. Accordingly, Benner invented a new non-standard nucleoside, 6-amino-5-nitro-3-(1′-beta-D-T-deoxyribofuranosyl)-2(1H)-pyridone (trivially designated as dZ when incorporated into sequences) to implement the pyDDA hydrogen bonding pattern. The nitro group rendered the otherwise electron-rich heterocycle stable against both oxidation and epimerization under standard conditions. When paired with a corresponding puAAD nucleotide, duplexes were formed with stabilities that, in many cases, were higher than those observed in comparable strands incorporating the dG:dC nucleobase pair. This invention is covered by U.S. Pat. No. 8,053,212, which is incorporated herein in its entirety by reference.
While Z supports binding of oligonucleotide analogs containing it to complementary strands that match a nucleobase implementing the puAAD hydrogen bond pattern, it was not clear that polymerases would accept this unnatural base pair. Polymerases are known to be idiosyncratic, meaning that experimentation is necessary to ascertain whether a specific implementation of a non-standard hydrogen bonding scheme can be accepted by a polymerase. This includes special architectures by which dZ:dP pairs might be synthesized in duplex oligonucleotides using various polymerases. These include PCR and nested PCR, termed “higher level PCR” architectures in U.S. patent application Ser. No. 12/999,138. These require thermal cycling to separate duplexes in each cycle of amplification.
Another architectures is known in the art as “rolling circle amplification” (RCA) [Dean, F. B., Nelson, J. R., Giesler, T. L., Lasken, R. S. (2001) Rapid amplification of plasmid and phage DNA using Phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Research 11, 1095-1099] [Johne, R., Mueller, H., Rector, A., can Ranst, M., Steven, H. (2009) Rolling-circle amplification of viral DNA genomes. Trends Microbiol. 17, 205-211.] using phi29 polymerase. These references are hereby incorporated herein in their entireties by reference.
In contrast to various PCR architectures, RCA does not require thermal cycling. Therefore, RCA does not require a thermostable polymerase. Rather, RCA uses a cyclic single stranded DNA molecule as a template. A primer is annealed to this cyclic single stranded DNA. Then, a polymerase that does strand displacement extends the primer to give a long single stranded product that is a concatamer of the segments that complement the circular template.
It is known in the art that dZ nucleotide incorporated into an oligonucleotide supports binding of oligonucleotides containing it to a complementary strand that incorporates at a matched position a nucleobase implementing the puAAD hydrogen bond pattern, it was not clear that polymerases would accept this unnatural base pair. Polymerases are known to be idiosyncratic [Horlacher, J., Hottiger, M., Podust, V. N., Huebscher, U., Benner, S. A. (1995) Expanding the genetic alphabet: Recognition by viral and cellular DNA polymerases of nucleosides bearing bases with non-standard hydrogen bonding patterns. Proc. Natl. Acad. Sci., 92, 6329-6333], meaning that experimentation is necessary to ascertain whether a specific implementation of a non-standard hydrogen bonding scheme can be accepted by a polymerase that is not a close homolog of a polymerase that has already been experimentally examined. In the applications for which priority is claimed, this is shown for thermostable polymerases of Family A and Family B. This disclosure reports data showing that the pair between dZ and dP is also formed in duplex DNA by strand-displacing polymerases used in a rolling circle polymerase synthesis.